The present invention relates to novel, stabilized insulin analogs and, in a preferred embodiment, to novel injectable solutions having prolonged insulin action.
In the treatment of diabetes mellitus, many varieties of insulin preparations have been suggested and used. Some of the preparations are fast acting and other preparations have more or less prolonged actions. Such a prolonged action may be obtained by administering the insulin as a suspension of insulin crystals. The crystalline preparations can be obtained by crystallization of insulin in the presence of zinc (such as LENTE.TM., see Schlichtkrull: Insulin Crystals, Chemical and Biological Studies on Insulin Crystals and Insulin Zinc Suspensions, Munksgaard, 1958) or by crystallization of insulin in the presence of zinc and protamine (such as NPH-insulin, see Rep.Steno Mem.Hosp. 1 (1946), 60).
Acid solution of insulin have been used earlier, both as short-acting preparations and as long-acting preparations containing protamine and zinc. However, the chemical stability of insulin at pH-values below 4.5 is low, as formation of desamidoinsulins (Sundby, F., J.Biol.Chem. 237 (1962), 3406-3411) and covalent dimers (Steiner et al, Diabetes 17 (1968), 725-736) takes place. In the pH-range 4.5-6.5, insulin precipitates. Hence, in order to make soluble short-acting insulin preparations (addition of blood-flow enhancing agent) and long-acting insulin preparations (addition of protamine and/or zinc) an insulin stable at low pH would be desirable.
It is known that during the acid ethanol extraction of mammalian insulins many dimers are formed (Steiner) and, furthermore, monodesamidoinsulins are formed under acid conditions (Sundby).
One disadvantage in the use of the known suspensions of zinc insulin crystals or of zinc protamine insulin is the necessity of shaking the vial in order to ensure that the correct amount of insulin is being injected and to ensure that the concentration of insulin in the vial remains constant throughout its use. In PENEILL .TM. cartridges where air must be absent, prolonged acting insulin suspensions require the incorporation of a solid body in the cartridge to enable agitation. The shaking of insulin suspensions and insulin solutions with air is in itself an undesirable process, as insulin has a tendency to denature under formation of fibrills at water-air interfaces. Consequently, solutions of insulins with prolonged action are desirable.
Solutions of insulin derivatives having a prolonged action were obtained from insulin that had been modified in its amino groups by reaction with phenylisocyanate (so-called Isoinsulin, see Hallas-Moeller: Chemical and Biological Insulin Studies based upon the Reaction between Insulin and Phenylisocyanate, Copenhagen 1945). Similarly, Al,B29-di-Boc substituted insulin (Boc designates tertiary butyloxycarbonyl) was reported to show a prolonged insulin action after subcutaneous administration (see Geiger & Enzmann in: Proinsulin, Insulin, C-peptide; Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide, Tokushima 1978; Amsterdam Oxford 1979, 306-310). The Al,B29-di-Boc substituted insulin was found to exhibit a too slightly prolonged action to be clinically useful.
Solutions of unmodified insulins require large amounts of zinc ions (for example, 0.4-1 mg/U insulin) in order to exhibit a prolonged action (see J.Pharmacol. 55 (1935), 206). Injection of such large doses of zinc ions will probably cause pain and such solutions have, therefore, never been used in therapy.
The isoelectric point of insulin is about 5.5 and attempts have been made to decrease the solubility of insulin derivatives at neutral pH by shifting the isoelectric point upwards, for example, through additions, in the N-terminus of the B-chain, of basic amino acids like lysine or arginine (see, for example, German Offenlegungsschrift No. 2,042,299) or with the basic dipeptide arginyl-arginine (see Geiger & Enzmann cited above). However, near its isoelectric point the solubility of Arg.sup.B(-1) -Arg.sup.BO insulin was much higher than that of the parent insulin.
Japanese patent application No. 55-144032 relates to analogues to human insulin wherein the B30-amino acid has been replaced by an amino acid having at least five carbon atoms, and amides and esters thereof. These insulin analogues were to be used in patients who had developed antibodies against mammalian insulins. In the Japanese patent application, six specific compounds are described, none of which were stated to have prolonged action. No specific injectable preparations are described in the Japanese patent application.
European patent application No. 84108442 relates to insulin analogues wherein a basic, organic group is attached to the B30-amino acid thereby introducing a positive charge at neutral pH. In these analogues, the B30-amino acid is neutral and, preferably, threonine as in human insulin. German patent application No. 3,327,709 relates to a suspension of crystals of the derivatives described in the above-noted European patent application as well as an aromatic hydroxy compound. German patent application No. 3,326,473 relates to a medicament containing a mixture of insulin compounds, of which at least one is described in the above-noted European patent application.
One object of this invention is to prepare insulin derivatives with improved properties.
A second object of this invention is to prepare insulin solutions having an improved stability.
A third object of this invention is to prepare insulin preparations with no or low immunogenic action.
A fourth object of this invention is to prepare insulin analogs which are dissolved at pH values below about 5.8.
The present invention arose within the context of prolonged action insulin described above but is not limited thereto.
Acid insulin solutions, a form that many years ago represented the only insulin form employed in diabetes therapy, are relatively unstable (with substantial deamidation at A-21 taking place). Substituting a more stable amino acid residue for Asn.sup.A21 improves the stability of the insulin molecule at pH levels lower than its isoelectric point. Solutions of the A-21 substituted insulin analogs of this invention are characterized by improved stability at acid pH levels.